Epigenetic misregulation is consistent with various non-Mendelian features of bipolar disorder (BPD), such as the discordance of identical twins, significant fluctuations in disease course, sex- and parental origin- effects, and the presence of familial and sporadic cases. To date, however, few studies have investigated the role of epigenetic factors in BPD and other psychiatric disease, and none have utilized an epigenome-wide approach. In our recent study, we used 12,192- feature containing CpG-island microarrays to identify DNA methylation changes in the prefrontal cortex of individuals affected with BPD and schizophrenia. In this study, we found evidence for psychosis-associated DNA methylation differences in several dozen loci. While this effort proved the principle that microarray-based DNA methylation profiling can reveal epigenetic changes in psychiatric patients, the first epigenome scan tested a small sample, explored only ~0.5% of the epigenome, and likely revealed only minor epigenetic changes, most of which are non-causal for major psychosis. This project is dedicated to a second, much more powerful and comprehensive epigenomic analysis of BPD. The main objective of this project is to identify major etiological epimutations in BPD. We will scan the entire non-repetitive genome in 1,000 DNA samples from BPD patients and controls using high density Affymetrix tiling microarrays. In comparison to the previous study, we have increased the sample of post-mortem brain tissues 3-fold (100 BPD patients and 100 controls). We will investigate DNA methylomes in neurons separately from the glial cells, which will help to estimate the confounding effects of cellular heterogeneity in the brain. In addition to the brain sample, a full microarray- based DNA methylation scan will be performed on peripheral blood leukocytes, buccal epithelial cells, and germline of BPD patients and controls. This effort should help i) to address cause-effect relationship between the detected epigenetic changes and BPD, ii) to identify heritable BPD epimutations, and iii) to uncover the networks of inter- dependence of epigenetic regulation of human genes across different tissues of BPD patients and control individuals. The project will generate ~10 billion microarray data points that will be processed using a battery of bioinformatics tools. The raw and processed data will be shared with the scientific community via our collaboration with the Epigenomics Data Analysis and Coordination Center (EDACC). The project should help us understand the molecular basis for the inherited and acquired nature of BPD, and further define the methodological guidelines for epigenomic studies in psychiatric research. The identification of epigenetic disease markers would be of critical importance in the diagnostic, treatment, and prophylactic applications in BPD. They may also bring new opportunities for the rational design of the next generation of drugs for BPD. Public Health Relevance: DNA Methylome Analysis in Bipolar Disorder Project Narrative Although there is a general agreement that bipolar disorder is caused by the interaction of damaged genes and hazardous environmental factors, identification of such etiological factors has been slow, despite significant research efforts. We performed a detailed re-analysis of the scientific literature on major psychiatric disease and concluded that a third group of factors, epigenetic factors, may disturb normal activity of the brain. The main role of epigenetic factors is to regulate gene activity, and this is achieved by either chemical changes in DNA or chromosomal proteins. We have accomplished the first pilot epigenome scan and detected a number of interesting epigenetic changes in the brains of psychiatric patients. In this application, we suggest to perform a second, much more comprehensive and informative epigenome scan in bipolar disorder. The plan is to investigate epigenetic status of the entire genome in 1,000 brains and other tissues of bipolar disease patients and control individuals. We will use cutting edge microarray technology that allows us to perform millions of measurements in one single experiment. We will put particular effort in differentiating causal epigenetic changes that increase the risk of developing bipolar disorder from the non-causal associations, i.e. epigenetic changes induced by the disease process, treatment or other factors. This research may lead to a better understanding of the changes in the regulation of genes and genomes that occur in bipolar disorder. Also, this effort may have a dramatic impact on our understanding of the molecular mechanisms of the disturbed brain and lead to new strategies in diagnostics and treatment of major psychiatric illness.